Expression of Thy 1 antigen in normal and neoplastic mammary cells of mice.

The expression of Thy 1.2 (theta C3H) antigen was measured on the membranes of normal and neoplastic RIII and C3H mammary cells. Competitive inhibition assays revealed that the average membrane content of Thy 1.2 in mammary tissues was about equal to that of lymph node cells. Higher percentages of Thy 1.2-positive cells than mammary tumor virus (MuMTV)-positive cells were observed by immunofluorescence, which suggested that not all the Thy 1.2-positive cells recovered from tumors were also MuMTV-positive. Established tissue culture cell lines C3H and RIII MT expressed lower levels of Thy 1.2 than did cells from mammary tumors. Treatment with the synthetic gluco-corticoid dexamethasone increased the average Thy 1.2 expression in cultured mammary tumor cells as well as the levels of RNA-directed DNA polymerase. Since the percentages of Thy 1.2-positive cells also were greater in steroid-treated cultures, while fewer cells were needed to absorb a standard amount of anti-Thy 1.2 activity, it was concluded that dexamethasone enhanced membrane Thy 1.2 expression as well as MuMTV production by the cultured cells.

Retarded growth of lymphoma in immunodepressed mice.

Lymphoma cells were inoculated iv into normal and immunodepressed mice. In most instances the immunodepressed animals survived longer than their normal counterparts. This was true both with athymic and thymectomized animals that had been lethally irradiated and reconstituted with syngeneic bone marrow. The route of administration was critical, since increased survival of immunodeficient animals was not observed when the lymphoma cells were injected ip.

Resistance to syngeneic lymphoma cells as a result of immunization with chemically modified allogeneic lymphoma cells in mice.

Immunization with trinitrophenyl (TNP)-derivatized allogeneic lymphoma cells resulted in significant immunity to poorly immunogenic syngeneic lymphoma cells. Neither TNP-treated nor X-irradiated syngeneic lymphoma cells were immunogenic under similar experimental conditions. Immunization with untreated allogeneic lymphoma cells produced only minimal levels of resistance to challenge with syngeneic lymphoma cells. The complete set of antigens responsible for the immunity was carried exclusively on transformed lymphocytes because allogeneic TNP-derivatized lymph node and thymus cells also did not immunize. The immunity was transferred to nonimmune inbred BALB/c and A/J mice by spleen cells from immune donors. The Winn assay was used to measure the antilymphoma immunity in vivo. When immune spleen cell-lymphoma mixtures were inoculated sc at a ratio of 1,000:1, nonimmune mice were completely protected. Reactivity of immune lymphocytes to syngeneic lymphoma cells was also demonstrated in vitro by the 51Cr-release method. Immunization with TNP-derivatized allogeneic lymphoma cells resulted in measurable immune resistance to inocula of viable syngeneic tumor cells in excess of 100 times the tumorigenic dose. Induction of immunity to syngeneic lymphoma cells strictly required that the immunizing cells be histoincompatible at the major H-2 locus and possess tumor-specific antigen(s). Maximum immune sensitivity was observed only after chemical modification of the immunizing allogeneic lymphoma cells.

Virus-dependent cytostatic activity to mammary tumor cells of lymphocytes from normal mice.

A cytostasis assay has been used to study the natural immunity of mice to murine mammary tumor virus (MTV). Spleen cells from adults of all strains tested were found to be cytostatic to a variety of MTV-positive mammary tumor cell lines. Newborn spleen cells were unreactive in the same cytostasis assay. The degree of reactivity to the target cells was greater in spleen cell preparations from low MTV expressors than from syngeneic, high MTV expressors. The cytostasis was specific, since MTV antigens prepared from gradient-purified whole MT virions significantly blocked the reaction. In addition, spleen cells were totally nonreactive to MTV-negative cell lines. Other types of lymphoid cells, such as lymph node cells as well as peritoneal macrophages, were highly cytostatic under similar conditions. Spleen cells from nude athymic donors were not cytostatic. Since depletion of splenic T-lymphocytes by use of anti-theta serum also did not significantly affect cytostasis, it was concluded that T-cells were required for initiation of immunity to MTV but that the effector cells were not theta positive. Blocking factors were found to exist in the sera of mammary tumor-bearing animals that prevented cytostasis by reactive spleen cells.

Changes in the mitogen response of lymphoid cells with progressive tumor growth.

The response of spleen and lymph node cells from tumor-bearing animals to a variety of mitogens was measured. It was found that progressive tumor growth diminished the reactivity of spleen cells to the mitogen phytohemagglutinin. Lymph node cells from the same animal were not consistently so affected. The reactivity of spleen cells from tumor-bearing donors to concanavalin A was somewhat depressed, while stimulation by bone marrow-derived lymphocyte mitogens such as endotoxin and pokeweed mitogen was in some cases enhanced. Theta antigen expression in tumor bearer spleens was found to decline steadily after 7 days of tumor growth. Cytological analysis revealed that the normal structure of the spleens of tumor animals was infiltrated by myeloid elements. The changes described occurred regardless of whether the tumors were chemically or virally induced. Excision of tumors after long periods of growth resulted in prompt return of splenic phytohemagglutinin sensitivity. The data suggests that loss or incapacitation of parts of the normal thymus-derived lymphocyte components may occur in the spleens of animals with progressive neoplastic growth.

Accelerated growth of mammary tumor cells in normal and athymic mice after treatment in vitro with dexamethasone.

The tumorigenicity of dexamethasone-treated tissue-cultured mammary tumor cells was compared to that of untreated mammary tumor cells. The dexamethasone-induced cells progressed faster in normal syngeneic animals than did untreated mammary tumor cells. The fact that the growth rate differential was also observed in athymic mice suggests that T-lymphocyte-mediated immunity was not a factor in the accelerated progression in vivo of the tumor cells that had been cultured in the presence of dexamethasone.

Alternatives to pristane priming for ascitic fluid and monoclonal antibody production.

A variety of agents were compared to pristane for priming mice prior to ascitic fluid production. Incomplete Freund's adjuvant was found to be as good as or better than pristane for priming before hybridoma cell inoculation. Complete adjuvant was also useful while priming with proteose-peptone; thioglycollate, corn oil or mineral oil elicited only minimum amounts of ascitic fluid after hybridoma injection.

Development and characterization of monoclonal antibodies with specificity for metallic radioisotope chelators linked to antibodies and other proteins.

Monoclonal antibodies with specificity for the diethylenetriaminepentaacetic acid (DTPA) portion of the GYK-DTPA (glycyl-tyrosyl-lysine-DTPA) linker structure used to chelate metals to immunoglobulins have been prepared. A significant proportion of these antibodies were lambda light chain isotype. Competition assays demonstrated that DTPA, rather than GYK, was the binding site of the antibodies tested. These monoclonal antibodies should be useful reagents for use in assays specific for the presence of the common linker structure used to chelate metallic radioisotopes to monoclonal antibodies. The antibodies tested did not discriminate between chelated and unchelated DTPA.

Antitumor monoclonal antibodies generated by immunization with mucins.

This report describes the development and characterization of monoclonal antibody EG2.3. Although produced from a fusion that used splenocytes from donor mice immune to bovine salivary mucin (BSM), EG2.3 bound selectively to a number of human tumor cell lines including colon adenocarcinoma LS174T. Therefore, EG2.3 was compared to B72.3, another mucin (TAG-72) binding monoclonal antibody that also binds to LS174T. Like B72.3, EG2.3 reacted with an epitope on TAG-72. However, these two MAbs differed in a number of ways. Treatment of mucin or TAG-72 with periodate did not reduce the binding of EG2.3 to either antigen. In contrast, B72.3 did not react with either periodate treated antigens. Removal of sialic acid from either BSM or TAG-72 compromised the reactivity of both EG2.3 and B72.3. It was concluded that the EG2.3 binding site was distinct from the carbohydrate structure detected by B72.3.

Mouse mammary tumor virus as a model for viral carcinogenesis.

Evidence is presented suggesting that genetic control may be the limiting factor in mammary tumor virus (MTV) positive mammary oncogenesis. Studies with hybrid mice involving high and low MTV-expressing murine strains suggest that expression of MTV in milk may not be as important in tumorigenesis as previously thought. The usefulness of the murine MTV model in the study of mammary malignancy is discussed.

Augmented immunogenicity of tumor cell membranes produced by surface budding viruses: parameters of optimal immunization.

Membranes prepared from tumor cells infected with surface budding viruses are much more immunogenic than membranes from uninfected tumor cells. Factors affecting immunization with membranes from virus-infected tumor cells were studied. Preparations made with influenza virus were clearly superior to those prepared with vesicular stomatitis virus (VSV). Membranes infected with VSV were maximally immunogenic at a dose equivalent to a 10% cell pack whereas influenza-virus-infected membranes were immunogenic at 1/100th of this dose. Subcutaneous inoculation was better than other routes of administration. Maximum protection against challenge with viable tumor cells was afforded by two inoculations of VSV-infected membranes spaced 3 days apart or a single inoculation with influenza-virus-infected membranes. Administration of membranes in complete Freund's adjuvant either had no effect of induced a slight degree of tumor enhancement. Immunization with influenza-virus-infected membranes significantly reduced tumor size and incidence even at a challenge dose of tumor cells which was 50 times the LD100.

Improved hemagglutination inhibition assay for mammary tumor virus antigen.

It is our experience that the use of sheep red blood cells pretreated with formaldehyde and pyruvaldehyde before sensitizing with viral antigen yields results that are comparable to that obtained by using the tannic acid method. The double aldehyde method provides the added advantage of being useful for assaying the viral antigen content in substances which contain high levels of interfering substances which would preclude the use of other assay methods.